Supplementary Figure 4: CD25⁻FOXP3^lo T_reg cell progenitors express TNFRSF in proportion to TCR signal strength and are responsive to TNFRSF costimulation

(a,b) CD25⁺Foxp3⁻ T_reg cell progenitors, and the alternately described population of T_reg cell progenitors which are CD25⁻Foxp3^lo are gated in red and blue, respectively, and are compared to conventional CD4SP (CD25⁻Foxp3⁻; gray shaded histogram) for expression of GITR and OX40. Raw values for GITR and Nur77-GFP from (c) CD25⁺Foxp3⁻ and (d) CD25⁻Foxp3^lo T_reg cell progenitors were plotted and used to calculate Pearson correlation coefficients. P-values assess whether the degree of correlation was statistically significant. (e) CD25⁻Foxp3^lo T_reg cell progenitors were sorted from Foxp3-RFP x Nur77-GFP reporter mice and incubated with 1 U/mL IL-2 and increasing concentrations of GITRL-Fc. The percentage of cells which upregulated CD25 and converted into mature CD25⁺Foxp3⁺ T_reg cells after 72h are shown in the scatter plot with a regression line applied. (f) The MFI of Nur77-GFP in newly formed CD25⁺Foxp3⁺ T_reg cells is shown after sorting CD25⁻Foxp3^lo T_reg cell progenitors from Foxp3-RFP x Nur77-GFP reporter mice and stimulating for 72h with 1 U/mL IL-2 and increasing concentrations of GITRL-Fc.